The progress of PD research is hindered by the lack of genetically engineered mouse models that develop PD-like progressive degeneration of nigrostriatal DA neurons in the midbrain. Several groups designed transgenic mice over-expressing WT, A53T, and A30P alpha-synuclein, but none of these mice show clear loss of midbrain DA neurons, which is likely resulted from a weaker expression of transgene in the nigrostriatal DA neurons. To overcome this technical challenge, we propose to develop a conditional alpha-synuclein transgenic mouse model in which &#945;-synuclein is designed to highly express in the midbrain DA neurons. To achieve this goal, we have developed a new line of tTA transgenic mice under the control of PITX3 promoter. PITX3, pituitary homeobox 3, is selectively expressed in midbrain DA neurons, including DA neurons in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). The expression of PITX3 starts from embryonic day 12.5 and stays through adulthood. Thus, our specific aims for this project are: Aim 1: To generate and characterize conditional alpha-synuclein WT and A53T transgenic mice under the control of PITX3 promoter; Aim 2: To investigate the cellular and molecular mechanisms of alpha-synuclein A53T mutation that cause dysfunction and degeneration of midbrain DA neurons. To selectively express alpha-synuclein A53T or LRRK2 G2019S mutation in nigrostriatal DA neurons, we have generated a new line of tetracycline transactivator (tTA) mice by inserting tTA coding sequence into the PITX3 gene locus via target DNA recombination approach. The tTA cDNA was introduced into the last coding exon of PITX3 gene and a copy of internal ribosomal entry site (IRES) sequence was inserted between the stop codon of PITX3 gene and the start codon of tTA gene. This genetic modification would lead to transcription of PITX3 and tTA bicistronic messenger RNA under the control of PITX3 gene locus. We have successfully developed two lines of PITX3-IRES-tTA knock-in mice. To determine the expression pattern of tTA in these animals, we crossed PITX3-IRES-tTA mice with a line of tetracycline operator-controlled GFP transgenic mice Tg (tetO-HIST1H2BJ/GFP) 47Efu/J. As expected, the expression of GFP was restricted to midbrain DA neurons in the double transgenic mice. We then crossed alpha-synuclein A53T mice with PITX3-IRES-tTA mice to selectively express this PD-linked dominant mutation in midbrain DA neurons. PITX3-IRES-tTA/A53T double transgenic mice were smaller and displayed decreased rearing activity as well as gait abnormalities at 1 month of age. As initially designed, the transgene was only expressed by midbrain DA neurons revealed by an antibody specific to human alpha-synuclein. The human alpha-synuclein immuno-reactivity was observed in the soma of midbrain DA neurons and at their axonal terminals ending at the striatum and other areas of the brain. Previous studies have demonstrated that dysfunction of nigrostriatal DA neurons plays a key role in the pathogenesis of PD. However, how the PD-related genetic mutations affect the function and survival of nigrostriatal DA neurons in vivo is not well studied. Here we have successfully developed novel lines of transgenic mice that selectively express PD-linked dominant genetic mutations in the midbrain DA neurons. Our preliminary data indicated that over-expression of alpha-synuclein A53T mutation in midbrain DA neurons caused a significant decrease of motor activities and obvious gait abnormalities at as early as 1 month of age. There was no apparent degeneration of DA neurons at 1 month of age, indicating a likely functional alteration of these neurons in regulating motor activities. We will keep monitoring the progression of motor behavioral and pathological changes of PITX3-IRES-tTA/A53T double transgenic mice at different ages. We will also apply neurochemical and electrophysiological studies on potential deficiency in dopamineric transmission in these mice. Gene expression array and cell biology studies on primary cultured midbrain DA neurons will help to define the molecular and cellular pathways leading to dysfunction and degeneration of DA neurons.